Cage amine ligands for metallo-radiopharmaceuticals

ABSTRACT

The present invention relates to compounds that are useful as metal ligands and which can be bound to a biological entity such as a molecular recognition moiety and methods of making these compounds. Once the compounds that are bound to a biological entity are coordinated with a suitable metallic radionuclide, the coordinated compounds are useful as radiopharmaceuticals in the areas of radiotherapy and diagnostic imaging. The invention therefore also relates to methods of diagnosis and therapy utilising the radiolabelled compounds of the invention.

RELATED APPLICATIONS

This application is a Division of U.S. patent application Ser. No.14/363,219, filed Jun. 5, 2014, which is a U.S. National Stage Filingunder U.S.C. 371 of International Patent Application Serial No.PCT/AU2012/001484, filed Dec. 6, 2012, and published on Jun. 13, 2013 asWO 2013/082656 A1, which claims the benefit of priority of U.S.Provisional Application Ser. No. 61/567,262, filed Dec. 6, 2011, each ofwhich are incorporated herein by reference in their entirety.

FIELD

The present invention relates to compounds that are useful as metalligands and which can be bound to a biological entity such as amolecular recognition moiety and methods of making these compounds. Oncethe compounds that are bound to a biological entity are coordinated witha suitable metallic radionuclide, the coordinated compounds are usefulas radiopharmaceuticals in the areas of radiotherapy and diagnosticimaging. The invention therefore also relates to methods of diagnosisand therapy utilising the radiolabelled compounds of the invention.

BACKGROUND

Radiolabelled compounds may be used as radiopharmaceuticals in a numberof applications such as in radiotherapy or diagnostic imaging. In orderfor a radiolabelled compound to be employed as a radiopharmaceuticalthere are a number of desirable properties that the compound shouldideally possess such as acceptable stability and, where possible, adegree of selectivity or targeting ability.

Initial work in the areas of radiopharmaceuticals focussed on simplemetal ligands which were generally readily accessible and hence easy toproduce. A difficulty with many of these radiolabelled compounds is thatthe complex formed between the ligand and the metal ion was notsufficiently strong and so dissociation of the metal ion from the ligandoccurred in the physiological environment. This was undesirable as withthe use of ligands of this type there was no ability to deliver theradiopharmaceutical to the desired target area in the body as metalexchange with metal ions in the physiological environment meant thatwhen the radiopharmaceutical compound arrived at the desired site ofaction the level of radiolabelled metal ion coordinated to the compoundhad become significantly reduced. In addition where this type ofexchange is observed the side effects experienced by the subject of theradiotherapy or radio-imaging are increased as radioactive material isdelivered to otherwise healthy tissue in the body rather thanpredominantly to its place of action.

In order to overcome the problem of metal dissociation in thephysiological environment a number of more complicated ligands have beendeveloped and studied over time. Thus, for example a wide range oftetra-azamacrocycles based on the cyclam and cyclen framework have beeninvestigated. Examples of ligands of this type include DOTA and TETA.

Unfortunately, even with these ligands there is still dissociation ofthe metal with certain derivatives. For example, some derivatives sufferfrom dissociation of Cu from the chelate as a consequence oftranschelation to biological ligands such as copper transport proteinseither as Cu²⁺ or following in vivo reduction to Cu⁺.

In order to increase the stability of radiolabelled compounds thereforehexaminemacrobicyclic cage amine ligands, known by their trivial namesarcophagines have been developed. These cage ligands form remarkablystable complexes with metals such as Cu²⁺ and have fast complexationkinetics even at low concentrations of metal at ambient temperatures.These features therefore make ligands of this type particularly wellsuited in radiopharmaceutical applications, especially thoseapplications involving copper.

Once the problem of stability of the complex between the ligand and themetal had been overcome attention turned to developing ways in which theligand could be functionalised to incorporate targeting molecules withinthe ligand without compromising the stability of the metal ligandcomplex or the ultimate biological activity of the targeting molecule. Anumber of different targeting molecules are known in the art and theissue became how best to attach these to the ligand molecules.

In general the targeting molecule (or molecular recognition moiety as itis sometimes known) is attached to the ligand to provide a finalcompound containing both a ligand and a molecular recognition moiety.Whilst these compounds may contain a single molecular recognition moietythey may also be multimeric constructs where the ligand is attached totwo (or more) molecular recognition moieties. This is typicallydesirable as a multimeric construct can possess higher affinity for atarget receptor than its monomeric equivalent. This is in part due to anincrease in the local concentration of the targeting group, allowing itto compete more effectively with endogenous ligands. In addition incircumstances where there is sufficient length between two or moretargeting groups within a multimeric construct, then cooperative bindingis possible, and two or more targeting groups will bind to two or morereceptor sites at the same time. Indeed it has been observed that invivo, a multimeric construct often demonstrates higher target tissueaccumulation than its monomeric equivalent. Without wishing to be boundby theory it is thought that this is due to the higher affinity of themultimeric construct for the target receptor than that of the monomericconstruct. Furthermore, the multimeric construct has a higher molecularweight than the monomeric construct and therefore prolongedbioavailability (as it is more resistant to degradation in thephysiological environment). This can result in increased accumulationand retention in target tissue.

Initial work in the caged ligand area looked at direct couplingreactions of the primary amines of the cage amine ‘diaminosarcophagine’,1,8-diamino-3,6,10,13,16,19-hexaaza bicyclo[6.6.6] icosane ((NH₂)₂sar),with peptides using standard coupling procedures. Unfortunately for avariety of reasons this has proven to be relatively inefficient and workin this area ceased. Workers then focussed on the incorporation of anaromatic amine to produce SarAr. The pendent aromatic amine can be usedin conjugation reactions with the carboxylate residues of peptides orantibodies and it has been shown that SarAr could be conjugated toanti-GD2 monoclonal antibody (14.G2a) and its chimeric derivative(ch14.8) and the conjugate has been radiolabelled with ⁶⁴Cu.

A difficulty with this approach is that in reaction of the aromaticamine in the conjugation step there are 8 other nitrogen atoms in theSarAr molecule that are available for competing reactions leading to thepotential for the creation of a large number of impurities that isundesirable from a pharmaceutical sense. Whilst these could potentiallybe overcome by the use of substantial protective group chemistry this isclearly undesirable from a synthetic standpoint and scale up on acommercial scale.

An alternative approach has been to elaborate the ligand to incorporatecarboxylate functional groups and incorporate peptides or antibodies viatheir N-terminal amine residues and this approach is of particularimportance when the C-terminus is crucial to biological activity.Studies have shown that (NH₂)₂sar, can be functionalised with up to fourcarboxymethyl substituents via alkylation reactions with chloroaceticacid and the introduced carboxymethyl arms can be used as a point offurther functionalisation and EDC-coupling reactions can then be used tointroduce amino acids.

Unfortunately a potential disadvantage of these systems is thatintramolecular cyclisation reactions can still occur in which thecarboxymethyl arm reacts with a secondary amine of the cage framework toform lactam rings resulting in quadridentate rather than sexidentateligands. Accordingly whilst this approach can be followed the potentialfor unwanted side reactions is clearly undesirable from a commercialperspective.

Further studies directed towards the functionalisation of ((NH₂)₂sar)was based around its reaction with activated di-carbonyl compounds suchas acid anhydrides leading to the formation of an amide bond to theamine nitrogen and a free carboxylic acid moiety which was available forfurther elaboration to the desired binding onto a molecular recognitionmoiety. This lead to the formation of a carbonyl moiety as the “stick”end on the cage ligand and this was not always easy to elaborate byattachment to the molecular recognition moiety as was optimal.Accordingly there was a desire to probe ways in which the cage ligandwith a pendant carboxylic acid moiety could be attached to a biologicalentity to make this step in the overall process more efficient.

SUMMARY

In one aspect there is provided a method of functionalising a compoundof the formula (1) or a metal complex thereof:

wherein Lig is a nitrogen containing macrocyclic metal ligand;

L is a bond or a linking moiety;

to modify its ability to bind to a biological entity the methodcomprising;

(a) converting the compound of formula (1) or a metal complex thereof toa compound of formula (2) or a metal complex thereof

wherein L¹ is a spacer group;

(b) converting the compound of formula (2) or a metal complex thereof toa compound of formula (3) or a metal complex thereof

wherein R is a moiety capable of binding to a biological entity or aprotected form thereof or a synthon thereof.

In the steps of the present method the conversions or reactions may becarried out on the compounds per se or their metal complexes. Whilst thereactions can be carried out on the uncomplexed compounds in manyinstances this is undesirable as nitrogen atoms in the nitrogencontaining macrocyclic metal ligand may interfere with the desiredreaction. As such by first forming the metal complex the metal acts tode-activate these nitrogens in the nitrogen containing macrocyclic metalligand and so acts as a pseudo protecting group for the ligand nitrogenatoms. As such in one embodiment it is desirable to carry out theconversions and reactions on the metal complex of the compound inquestion. A number of metals may be used for this purpose with magnesiumbeing found to be particularly suitable.

By elaborating the compound of formula (1) above using the methodoutlined a large number of functionalised metal chelating ligands can beproduced. Accordingly in an even further aspect the present inventionprovides a compound of the formula (3):

wherein Lig is a nitrogen containing macrocyclic metal ligand;

L is a bond or a linking moiety;

L¹ is a spacer group;

R is a moiety capable of binding to a biological entity or a protectedform thereof or a synthon thereof;

As with any group of structurally related compounds which possess aparticular utility, and methods for their production, certainembodiments of variables of the compounds of the formula (3) which areparticularly useful in their end use application.

In the compounds of formula (3) the L moiety serves as a linking moietythat serves to act as a spacer between the two carbonyl moieties whichseparate the ligand which can be bound to the radionuclide and the pointof further elaboration. As such whilst it is desirable that there be acertain degree of separation between the two in order to ensure that thetwo entities do not interfere with each other's activity it is alsoimportant that the two are not so far removed such that the radionuclideis not effectively delivered to its site of operation.

In some embodiments L is a linking moiety having from 1 to 20 atoms inthe normal chain. In some embodiments L is a linking moiety having from1 to 15 atoms in the normal chain. In some embodiments L is a linkingmoiety having from 1 to 12 atoms in the normal chain. In someembodiments L is a linking moiety having from 1 to 10 atoms in thenormal chain. In some embodiments L is a linking moiety having from 1 to8 atoms in the normal chain. In some embodiments L has 8 atoms in thenormal chain. In some embodiments L has 7 atoms in the normal chain. Insome embodiments L has 6 atoms in the normal chain. In some embodimentsL has 5 atoms in the normal chain. In some embodiments L has 4 atoms inthe normal chain. In some embodiments L has 3 atoms in the normal chain.In some embodiments L has 2 atoms in the normal chain. In someembodiments L has 1 atom in the normal chain.

A wide range of possible moieties may be use to create a linking moietyof this type. Examples of suitable moieties that may be used in thecreation of L include optionally substituted C₁-C₁₂alkyl, substitutedC₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl.

In some embodiments L is a group of the formula:

—(CH₂)_(q)CO(AA)_(r)NH(CH₂)₅—

wherein each AA is independently an amino acid group;

q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, and 8;

r is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8;

s is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5,6, 7, and 8.

In some embodiments q is 1. In some embodiments q is 2. In someembodiments q is 3. In some embodiments q is 4. In some embodiments q is5. In some embodiments q is 6. In some embodiments q is 7. In someembodiments q is 8.

In some embodiments r is 0. In some embodiments r is 1. In someembodiments r is 2. In some embodiments r is 3. In some embodiments r is4. In some embodiments r is 5. In some embodiments r is 6. In someembodiments r is 7. In some embodiments r is 8.

In some embodiments s is 0. In some embodiments s is 1. In someembodiments s is 2. In some embodiments s is 3. In some embodiments s is4. In some embodiments s is 5. In some embodiments s is 6. In someembodiments s is 7. In some embodiments s is 8.

In some embodiments the amino acid is a naturally occurring amino acid.In some embodiments the amino acid is a non-naturally occurring aminoacid. In some embodiments the amino acid is selected from the groupconsisting of phenyl alanine, tyrosine, amino hexanoic acid andcysteine.

In some embodiments q is 3, r is o and s is 5. In these embodiments X isa group of the formula:

—(CH₂)₃CONH(CH₂)₅—

In some embodiments L is a group of the formula:

—(CH₂)_(a)—,

wherein optionally one or more of the CH₂ groups may be independentlyreplaced by a heteroatomic group selected from S, O, P and NR⁴ where R⁴is selected from the group consisting of H, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl;

a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 and 15.

In some forms of these embodiments the L group may contain a poly ethoxygroup (PEG). In some embodiments L is a group of the formula:

—(CH₂)_(l)—(CH₂CH₂O)_(m)(CH₂)_(n)—

wherein l is an integer selected from the group consisting of 0, 1, 2,3, 4, 5, 6, 7, 8, 9, and 10;

wherein m is an integer selected from the group consisting of 0, 1, 2,3, 4, 5, 6, 7, 8, 9, and 10; and

wherein n is an integer selected from the group consisting of 0, 1, 2,3, 4, 5, 6, 7, 8, 9, and 10.

In some embodiments l is selected from the group consisting of 0, 1, 2,3, 4, and 5. In some embodiments l is 5. In some embodiments l is 4. Insome embodiments l is 3. In some embodiments l is 2. In some embodimentsl is 1. In some embodiments l is 0.

In some embodiments m is selected from the group consisting of 0, 1, 2,3, 4, and 5. In some embodiments m is 5. In some embodiments m is 4. Insome embodiments m is 3. In some embodiments m is 2. In some embodimentsm is 1. In some embodiments m is 0.

In some embodiments n is selected from the group consisting of 0, 1, 2,3, 4, and 5. In some embodiments n is 5. In some embodiments n is 4. Insome embodiments n is 3. In some embodiments m is 2. In some embodimentsn is 1. In some embodiments n is 0.

Specific examples of L groups of this type include—CH₂—(CH₂CH₂O)₃(CH₂)₃—; and —(CH₂CH₂O)₃(CH₂)₂—. As will be appreciatedby a skilled worker in the field the values of l, m and n can be variedwidely to arrive at a large number of possible L groups of this type.

In some embodiments L is a group of the formula:

—(CH₂)_(a)—,

wherein optionally one or more of the CH₂ groups may be independentlyreplaced by a heteroatomic group selected from S, O, P and NR⁴ where R⁴is selected from the group consisting of H, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl; and

a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9 and 10.

In some embodiments a is selected from the group consisting of 1, 2, 3,4, and 5. In some embodiments a is 4. In some embodiments a is 3. Insome embodiments a is 2. In some embodiments a is 1.

In some embodiments L is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂— and —CH₂OCH₂—.

In some embodiments L is —(CH₂)—. In some embodiments L is —(CH₂)₂—. Insome embodiments L is —(CH₂)₃—. In some embodiments L is —(CH₂)₄—. Insome embodiments L is —(CH₂)₅—. In some embodiments L is —(CH₂)₆—. Insome embodiments L is —(CH₂)₇—. In some embodiments L is —(CH₂)₈—. Insome embodiments L is —(CH₂)₉—. In some embodiments L is —(CH₂)₁₀—.

In some embodiments the ligand (Lig) may be a tetra-azamacrocycle basedon the cyclam and cyclen framework. In some embodiments Lig is anitrogen containing cage metal ligand. Cage ligands of this type aretypically useful as they bind strongly to metal ions leading to a stablecomplex being formed.

In some embodiments Lig is a nitrogen containing cage metal ligand ofthe formula:

V is selected from the group consisting of N and CR¹;

each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO;

each p is independently an integer selected from the group consisting of2, 3, and 4;

R¹ is selected from the group consisting of H, OH, halogen, NO₂, NH₂,optionally substituted C₁-C₁₂alkyl, optionally substituted C₆-C₁₈aryl,cyano, CO₂R², NHR³, N(R³)₂ and a group of the formula:

L is as defined above;

R⁶ is selected from the group consisting of H, OH, NH₂, NHR³, N(R³)₂ andNH-L¹-R,

L¹ is as defined above;

R is a moiety capable of binding to a biological entity or a protectedform thereof or a synthon thereof;

R² is selected from the group consisting of H, halogen, an oxygenprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

each R³ is independently selected from the group consisting of H, L-R′,a nitrogen protecting group, optionally substituted C₁-C₁₂alkyl,—(C═O)-substituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl.

wherein L is as defined above;

R′ is H, optionally substituted C₁-C₁₂alkyl, or a moiety capable ofbinding to a biological entity.

In some embodiments Lig is a nitrogen containing cage metal ligand ofthe formula:

V is selected from the group consisting of N and CR¹;

each R^(x) and R^(y) are independently selected from the groupconsisting of H, CH₃, CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, ON, CONH₂ and CHO;

each p is independently an integer selected from the group consisting of2, 3, and 4;

R¹ is selected from the group consisting of H, OH, halogen, NO₂, NH₂,optionally substituted C₁-C₁₂alkyl, optionally substituted C₆-C₁₈aryl,cyano, CO₂R², NHR³, N(R³)₂;

R² is selected from the group consisting of H, halogen, an oxygenprotecting group, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl andoptionally substituted C₂-C₁₂ heteroalkyl;

each R³ is independently selected from the group consisting of H, L-R′,a nitrogen protecting group, optionally substituted C₁-C₁₂alkyl,—(C═O)-substituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl;

wherein L is as defined above and R′ is H, optionally substitutedC₁-C₁₂alkyl, or a moiety capable of binding to a biological entity.

In some embodiments Lig is a macrocyclic metal ligand of the formula:

wherein R^(x), R^(y) and p are as defined above.

In some embodiments Lig is a macrocyclic ligand of the formula:

wherein R^(x), R^(y), R′ and p are as defined above.

In some embodiments Lig is selected from the group consisting of:

wherein R¹ is as defined above.

In some embodiments Lig is a group of the formula:

In some embodiments L¹ is a linking moiety having from 1 to 20 atoms inthe normal chain.

In some embodiments L¹ is a group of the formula

—(CH₂)_(a)—,

wherein optionally one or more of the CH₂ groups may be independentlyreplaced by a heteroatomic group selected from S, O, P and NR⁴ where R⁴is selected from the group consisting of H, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl;

a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9 and 10.

In some embodiments a is selected from the group consisting of 1, 2, 3,4, and 5. In some embodiments a is 4. In some embodiments a is 3. Insome embodiments a is 2. In some embodiments a is 1.

In some embodiments L¹ is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, and —CH₂CH₂CH₂CH₂. In some embodiments L¹ is—CH₂CH₂CH₂—.

R is a moiety capable of binding to a biological entity, or a protectedform thereof or a synthon thereof. The moiety may have the ability tobind to a biological moiety such as an antibody, a protein, a peptide, acarbohydrate, a nucleic acid, an oligonucleotide, an oligosaccharide anda liposome or a fragment or derivative thereof.

As such the R group reacts or binds to a complementary moiety on thebiological entity of interest. For example in one embodiment the Rmoiety is a moiety capable of taking part in a click chemistry reactionwith a complementary moiety on a biological entity. Examples ofcomplementary paired functional groups that are well known to undergo“click” chemistry reactions are alkyne-azide, alkyne-nitrile oxide,nitrile-azide and maleimide-anthracene. Each of the paired complementaryfunctional groups gives rise to cyclic moieties when they directly reactwith one another in a covalent cycloaddition reaction. The personskilled in the art would be able to select other functional grouppairings capable of participating in cycloaddition reactions of thistype that satisfy the requirements of click chemistry. In generalthereof the identity of the R group will be chosen based on the relevantcomplementary R group on the biological entity of interest.

The R group may also react or bind to the biological entity by reactionwith a pendant moiety on the biological entity (either naturally presentor by modification of the biological entity). Once again a skilledworked in the field will be able to review the biological entity ofinterest in any specific circumstance and determine a suitable R groupto bind to the pendant moieties on the R group.

In some embodiments R is selected from the group consisting of —NCS,CO₂H, NH₂, an azide, an alkyne, an isonitrile, a tetrazine, maleimide,or a protected form thereof or a synthon thereof.

In some embodiments of the compounds of the invention the nitrogencontaining macrocyclic metal ligand is complexed with a metal ion. Theligand may be complexed with any suitable metal ion and may be used todeliver a range of metal ions. In some embodiments the metal in themetal ion is selected from the group consisting of Cu, Tc, Gd, Ga, In,Co, Re, Fe, Mg, Ca, Au, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt, Zn, Cd,Mn, Ru, Pd, Hg, and Ti.

In some embodiments the metal in the metal ion is a radionuclideselected from the group consisting of Cu, Tc, Ga, Co, In, Fe, and Ti.The present compounds have been found to be particularly applicableuseful in binding copper ions. In some embodiments the metal in themetal ion is a radionuclide selected from the group consisting of ⁶⁰Cu,⁶²Cu, ⁶⁴Cu and ⁶⁷Cu. In some embodiments the metal in the metal ion is⁶⁰Cu. In some embodiments the metal in the metal ion is ⁶²Cu. In someembodiments the metal in the metal ion is ⁶⁴Cu. In some embodiments themetal in the metal ion is ⁶⁷Cu.

The invention also relates to pharmaceutical compositions including acompound of the invention as described above and a pharmaceuticallyacceptable carrier, diluent or excipient.

In one embodiment of the methods of the invention step (a) comprises thesteps of:

(a1) converting the compound of formula (1) or a metal complex thereofinto a compound of formula (Ia) or a metal complex thereof:

wherein Lv is a group that can be displaced by a nitrogen moiety in anucleophillic substitution reaction;(a2) reacting the compound of formula (1a) or a metal complex thereofwith a nitrogen nucleophile of the formula:

to form a compound of formula (2) or a metal complex thereof.

In some embodiments of the method the group Lv is a leaving group. Insome embodiments steps (1a) and (1b) are carried out on the metalcomplexes of the respective compounds being reacted as the metal acts asa protecting group for the nitrogen atoms in the cage ligand.

In some embodiments the compound of formula (2) is converted to acompound of formula (3) by reacting the amine with a reagent selectedfrom the group consisting of an azide, thiosphosgene, carbon disulphideand an acid anhydride. In some embodiments this reaction is carried outon the metal complex of the compound of formula (2). In some embodimentsthis reaction is carried out on the non-complexed or free compound offormula (2). In embodiments where this occurs in which the compound offormula (2) is produced as a metal complex the method includes anadditional step of removing the metal complex prior to further reaction.

In some embodiments the reagent is an azide. In some embodiments thereagent is thiosphosgene. In some embodiments the reagent is carbondisulphide. In some embodiments the reagent is maleic anhydride.

These and other features of the present teachings are set forth herein.

DETAILED DESCRIPTION

In this specification a number of terms are used which are well known toa skilled addressee. Nevertheless for the purposes of clarity a numberof terms will be defined.

As used herein, the term “unsubstituted” means that there is nosubstituent or that the only substituents are hydrogen.

The term “optionally substituted” as used throughout the specificationdenotes that the group may or may not be further substituted or fused(so as to form a condensed polycyclic system), with one or morenon-hydrogen substituent groups. In certain embodiments the substituentgroups are one or more groups independently selected from the groupconsisting of halogen, ═O, ═S, —CN, —NO₂, —CF₃, —OCF₃, alkyl, alkenyl,alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl,cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl,cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl,cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl,heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl,arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy,alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl,alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl,alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy,heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy,heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl,arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl,arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl,aminosulfinylaminoalkyl, —C(═O)OH, —C(═O)R^(a), —C(═O)OR^(a),C(═O)NR^(a)R^(b), C(═NOH)R^(a), C(═NR^(a))NR^(b)R^(c), NR^(a)R^(b),NR^(a)C(═O)R^(b), NR^(a)C(═O)OR^(b), NR^(a)C(═O)NR^(b)R^(c),NR^(a)C(═NR^(b))NR^(c)R^(d), NR^(a)SO₂R^(b), —SR^(a), SO₂NR^(a)R^(b),—OR^(a), OC(═O)NR^(a)R^(b), OC(═O)R^(a) and acyl,

wherein R^(a), R^(b), R^(C) and R^(d) are each independently selectedfrom the group consisting of H, C₁-C₁₂alkyl, C₁-C₁₂haloalkyl,C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-C₁₀heteroalkyl, C₃-C₁₂cycloalkyl,C₃-C₁₂cycloalkenyl, C₂-C₁₂heterocycloalkyl, C₂-C₁₂heterocycloalkenyl,C₆-C₁₈aryl, C₁-C₁₈heteroaryl, and acyl, or any two or more of R^(a),R^(b), R^(C) and R^(d), when taken together with the atoms to which theyare attached form a heterocyclic ring system with 3 to 12 ring atoms.

In some embodiments each optional substituent is independently selectedfrom the group consisting of: halogen, ═O, ═S, —CN, —NO₂, —CF₃, —OCF₃,alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy,alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy,cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy,heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl,heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl,arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl,aminoalkyl, —COOH, —SH, and acyl.

Examples of particularly suitable optional substituents include F, Cl,Br, I, CH₃, CH₂CH₃, OH, OCH₃, CF₃, OCF₃, NO₂, NH₂, and CN.

As used herein the term “amino acid” refers to a molecule which containsboth an amine and a carboxyl function. The amino acid may be a naturalor an unnatural amino acid.

“Alkenyl” as a group or part of a group denotes an aliphatic hydrocarbongroup containing at least one carbon-carbon double bond and which may bestraight or branched preferably having 2-12 carbon atoms, morepreferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in thenormal chain. The group may contain a plurality of double bonds in thenormal chain and the orientation about each is independently E or Z.Exemplary alkenyl groups include, but are not limited to, ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. Thegroup may be a terminal group or a bridging group.

“Alkyl” as a group or part of a group refers to a straight or branchedaliphatic hydrocarbon group, preferably a C₁-C₁₂alkyl, more preferably aC₁-C₁₀alkyl, most preferably C₁-C₆ unless otherwise noted. Examples ofsuitable straight and branched C₁-C₆alkyl substituents include methyl,ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and thelike. The group may be a terminal group or a bridging group.

“Alkynyl” as a group or part of a group means an aliphatic hydrocarbongroup containing a carbon-carbon triple bond and which may be straightor branched preferably having from 2-12 carbon atoms, more preferably2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.Exemplary structures include, but are not limited to, ethynyl andpropynyl. The group may be a terminal group or a bridging group.

“Aryl” as a group or part of a group denotes (i) an optionallysubstituted monocyclic, or fused polycyclic, aromatic carbocycle (ringstructure having ring atoms that are all carbon) preferably having from5 to 12 atoms per ring. Examples of aryl groups include phenyl,naphthyl, and the like; (ii) an optionally substituted partiallysaturated bicyclic aromatic carbocyclic moiety in which a phenyl and aC₅-7cycloalkyl or C₅₋₇cycloalkenyl group are fused together to form acyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. Thegroup may be a terminal group or a bridging group. Typically an arylgroup is a C₆-C₁₈aryl group.

“Cycloalkyl” refers to a saturated monocyclic or fused or spiropolycyclic, carbocycle preferably containing from 3 to 9 carbons perring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and thelike, unless otherwise specified. It includes monocyclic systems such ascyclopropyl and cyclohexyl, bicyclic systems such as decalin, andpolycyclic systems such as adamantane. A cycloalkyl group typically is aC₃-C₉cycloalkyl group. The group may be a terminal group or a bridginggroup.

“Halogen” represents chlorine, fluorine, bromine or iodine.

“Heteroalkyl” refers to a straight- or branched-chain alkyl grouppreferably having from 2 to 12 carbons, more preferably 2 to 6 carbonsin the chain, in which one or more of the carbon atoms (and anyassociated hydrogen atoms) are each independently replaced by aheteroatomic group selected from S, O, P and NR′ where R′ is selectedfrom the group consisting of H, optionally substituted C₁-C₁₂alkyl,optionally substituted C₃-C₁₂cycloalkyl, optionally substitutedC₆-C₁₈aryl, and optionally substituted C₁-C₁₈heteroaryl. Exemplaryheteroalkyls include alkyl ethers, secondary and tertiary alkyl amines,amides, alkyl sulfides, and the like. Examples of heteroalkyl alsoinclude hydroxyC₁-C₆alkyl, C₁-C₆alkyloxyC₁-C₆alkyl, aminoC₁-C₆alkyl,C₁-C₆alkylaminoC₁-C₆alkyl, and di(C₁-C₆alkyl)aminoC₁-C₆alkyl. The groupmay be a terminal group or a bridging group.

“Heteroaryl” either alone or part of a group refers to groups containingan aromatic ring (preferably a 5 or 6 membered aromatic ring) having oneor more heteroatoms as ring atoms in the aromatic ring with theremainder of the ring atoms being carbon atoms. Suitable heteroatomsinclude nitrogen, oxygen and sulphur. Examples of heteroaryl includethiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole,benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan,isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole,pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole,isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine,naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine,acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole,isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-,or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3-indolyl, and2-, or 3-thienyl. A heteroaryl group is typically a C₁-C₁₈heteroarylgroup. The group may be a terminal group or a bridging group.

A “leaving group” is a chemical group that is readily displaced by adesired incoming chemical moiety. Accordingly in any situation thechoice of leaving group will depend upon the ability of the particulargroup to be displaced by the incoming chemical moiety. Suitable leavinggroups are well known in the art, see for example “Advanced OrganicChemistry” Jerry March 4^(th) Edn. pp 351-357, Oak Wick and Sons NY(1997). Examples of suitable leaving groups include, but are not limitedto, halogen, alkoxy (such as ethoxy, methoxy), sulphonyloxy, optionallysubstituted arylsulfonyl. Specific examples include chloro, iodo, bromo,fluoro, ethoxy, methoxy, methansulphonyl, triflate and the like.

The term “normal chain” refers to the direct chain joining the two endsof a linking moiety.

The term “pharmaceutically acceptable salts” refers to salts that retainthe desired biological activity of the above-identified compounds, andinclude pharmaceutically acceptable acid addition salts and baseaddition salts. Suitable pharmaceutically acceptable acid addition saltsof compounds of Formula (I) may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids are hydrochloric,sulfuric, and phosphoric acid. Appropriate organic acids may be selectedfrom aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic andsulfonic classes of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Additionalinformation on pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co.,Easton, Pa. 1995. In the case of agents that are solids, it isunderstood by those skilled in the art that the inventive compounds,agents and salts may exist in different crystalline or polymorphicforms, all of which are intended to be within the scope of the presentinvention and specified formulae.

The term “therapeutically effective amount” or “effective amount” is anamount sufficient to effect beneficial or desired clinical results. Aneffective amount can be administered in one or more administrations. Aneffective amount is typically sufficient to palliate, ameliorate,stabilize, reverse, slow or delay the progression of the disease state.An effective amount for radioimaging is typically sufficient to identifythe radionuclide in the subject.

The term “molecular recognition moiety” refers to an entity capable ofbinding to a particular molecular entity, typically a receptor locationin the physiological environment. The term includes antibodies,proteins, peptides, carbohydrates, nucleic acids, oligonucleotides,oligosaccharides and liposomes.

The term “oxygen protecting group” means a group that can prevent theoxygen moiety reacting during further derivatisation of the protectedcompound and which can be readily removed when desired. In oneembodiment the protecting group is removable in the physiological stateby natural metabolic processes. Examples of oxygen protecting groupsinclude acyl groups (such as acetyl), ethers (such as methoxy methylether (MOM), R-methoxy ethoxy methyl ether (MEM), p-methoxy benzyl ether(PMB), methylthio methyl ether, Pivaloyl (Piv), Tetrahydropyran (THP)),andsilyl ethers (such as Trimethylsilyl (TMS) tert-butyl dimethyl silyl(TBDMS) and triisopropylsilyl (TIPS).

The term “nitrogen protecting group” means a group that can prevent thenitrogen moiety reacting during further derivatisation of the protectedcompound and which can be readily removed when desired. In oneembodiment the protecting group is removable in the physiological stateby natural metabolic processes and in essence the protected compound isacting as a prodrug for the active unprotected species.

Examples of suitable nitrogen protecting groups that may be used includeformyl, trityl, phthalimido, acetyl, trichloroacetyl, chloroacetyl,bromoacetyl, iodoacetyl; urethane-type blocking groups such asbenzyloxycarbonyl (‘CBz’), 4-phenylbenzyloxycarbonyl,2-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,4-cyanobenzyloxycarbonyl, t-butoxycarbonyl (‘tBoc’),2-(4-xenyl)-isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl,1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl,2-(p-toluyl)-prop-2-yloxy-carbonyl, cyclo-pentanyloxy-carbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluylsulfono)-ethoxycarbonyl, 2-(methylsulfono)ethoxycarbonyl,2-(triphenylphosphino)-ethoxycarbonyl, fluorenylmethoxycarbonyl(“FMOC”), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 5-benzisoxalymethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,2-ethynyl-2-propoxycarbonyl, cyclopropylmethoxycarbonyl,4-(decycloxy)benzyloxycarbonyl, isobornyloxycarbonyl,1-piperidyloxycarbonlyl and the like; benzoylmethylsulfono group,2-nitrophenylsulfenyl, diphenylphosphine oxide, and the like. The actualnitrogen protecting group employed is not critical so long as thederivatised nitrogen group is stable to the condition of subsequentreaction(s) and can be selectively removed as required withoutsubstantially disrupting the remainder of the molecule including anyother nitrogen protecting group(s). Further examples of these groups arefound in: Greene, T. W. and Wuts, P. G. M., Protective Groups in OrganicSynthesis, Second edition; Wiley-Interscience: 1991; Chapter 7; McOmie,J. F. W. (ed.), Protective Groups in Organic Chemistry, Plenum Press,1973; and Kocienski, P. J., Protecting Groups, Second Edition, TheimeMedical Pub., 2000.

The term ‘click chemistry’ is used to describe covalent reactions withhigh reaction yields that can be performed under extremely mildconditions. A number of ‘click’ reactions involve a cycloadditionreaction between appropriate functional groups to generate a stablecyclic structure. The most well documented click reaction is the Cu(I)catalyzed variant of the Huisgen 1,3-dipolar cycloaddition of azides andalkynes to form 1,2,3-triazoles. Many click reactions arethermodynamically driven, leading to fast reaction times, high productyields and high selectivity in the reaction.

The compounds of the invention as discussed above may include a widevariety of nitrogen containing macrocyclic metal ligands.

The ligand may be a monocyclic nitrogen containing metal ligand based onthe cyclam or cyclen frameworks. Ligand of this type and derivativesthereof may be synthesised using methodology available in the art suchas in Bernhardt (J. Chem. Soc., Dalton Transactions, 1996, pages4319-4324), Bernhardt et al (J. Chem. Soc., Dalton Transactions, 1996,pages 4325-4330), and Bernhardt and Sharpe (Inorg Chem, 2000, 39, pages2020-2025). Various other ligands of this general type may be made byvariation of the procedures described in these articles.

The ligand may also be a cage like cryptand ligand as described forexample in Geue (Chemical communications, 1994, page 667). Cryptandligands of this type are described in U.S. Pat. No. 4,497,737 in thename of Sargeson et al, the disclosure of which is incorporated hereinby reference.

The synthesis involves a metal ion template reaction and involvescondensation of a tris-(diamine) metal ion complex (see column 3 lines30 to 35) with formaldehyde and an appropriate nucleophile in thepresence of base. The identity of the nucleophile will determine theidentity of the substituents on the cage ligand and a skilled addresseecan access a wide variety of substitution patterns around the cageligand by judicious choice of the appropriate amine used in thecondensation as well as the identity of the nucleophile.

In order to produce the compounds of formula (1) which are the staringmaterial for the present invention the amino substituted ligand or ametal complexed form thereof is reacted with an appropriate dicarbonylcompound under suitable reaction conditions to arrive at the finalproduct.

Whilst the reaction may be performed on the free ligand there is still apossibility of the reaction being compromised by the presence of thering nitrogen(s). As such it is desirable to perform the reaction usinga metal complex thereof as the metal serves to act as a protecting groupfor the secondary nitrogen atoms in the ring. Whilst this can beconducted using a number of different metals it is found that magnesiumis particularly suitable.

The reaction may be carried out in any suitable solvent which is inertto the two reactants with the identity of the solvent being determinedby the relative solubilities of the anhydride and the amine substitutedmetal ligand. Examples of solvents that may be used include aliphatic,aromatic, or halogenated hydrocarbons such as benzene, toluene, xylenes;chlorobenzene, chloroform, methylene chloride, ethylene chloride; ethersand ethereal compounds such as dialkyl ether, ethylene glycol mono or-dialkyl ether, THF, dioxane; nitriles such as acetonitrile or2-methoxypropionitrile; N, N-dialkylated amides such asdimethylformamide; and dimethyl acetamide, dimethylsulphoxide,tetramethylurea; as well as mixtures of these solvents with each other.

The reaction may be carried out at any of a number of suitabletemperatures with the reaction temperature being able to be readilydetermined on a case by case basis. Nevertheless the reactiontemperature is typically carried out at from 0 to 100° C., moretypically 50 to 80° C.

The reaction may be carried out using a wide variety of activateddicarbonyl compounds. In some embodiments the activated dicarbonylcompound is an anhydride of the formula:

wherein L is as defined above and Z is O, S or NR².

Anhydride compounds of this type are generally readily available forcertain values of L and thus these compounds may be readily used forvalues of L for which they are obtainable. It is desirable that they beutilised where possible as the potential for side reactions is reducedsomewhat with these compounds.

In some embodiments the activated dicarbonyl compound is a compound ofthe formula:

wherein L is as defined above. Y is OH or a protected from thereof andLv is a leaving group. The Lv group on the compounds of this type may beany suitable leaving group but is typically selected from the groupconsisting of Cl, Br, CH₃SO₃, CH₃C₆H₄SO₃, and a group of the formula:

In choosing a suitable leaving group for reactions of this type theskilled worker in the art will have regard for the functionality of theremainder of the molecule and the ease of production of the activateddicarbonyl compound in each instance.

The reaction is also typically carried out in the presence of a base asthis is found to facilitate the reaction. Examples of suitable basesinclude hindered tertiary amines with trialkyl amines such astrimethylamine, triethyleneamine, diisopropylethyl amine being suitableexamples of bases for use in the reaction. The amount of base used issuch that it is in a significant molar excess so as to ensure that thereaction does not become affected by acidification as it progresses.

The exact compound produced will depend upon the reaction stoichiometryand the starting materials with a skilled addressee being able to adjusteither of these variables to produce the desired final product.

In addition it is desired that the linker L be extended to besignificantly longer than the compounds readily accessible by the routedetailed above it is possible to elaborate the carboxy group (such as bystandard peptide chemistry techniques) to introduce further amino acidgroups to the chain. The methods of achieving reactions of this type arewell within the skill of a skilled addressee in the area.

In the method of the invention the compounds of formula (1) areconverted to compounds of formula (2). This conversion may be carriedout in any way known in the art and may be carried out as a single stepprocess or as a multi-step process.

In addition depending upon the substituents on the Lig group of thecompounds of formula (1) it may be necessary to protect the substituentsfrom interfering with the reaction. For example the applicants havefound that where the Lig group contains a free amino group (such as willbe the case if the original ligand was 1,8-diamino-Sar) and then it isdesirable to first protect the amino group prior to reaction with asuitable nitrogen protecting group. An example of a suitable protectinggroup of this type is the acetyl group.

As stated previously the conversion may be a one step or a multi-stepprocess. Thus for example an amide formation reaction could be carriedout (typically in the presence of a coupling reagent) to form an amidebond by reaction of compound of formula (1) with a compound of theformula NH₂L₁NH₂. It has been found, however, that in many instances itis desirable to carry out the conversion as a multi-step process byfirst converting the carboxylic acid moiety to an activated form thereof

Accordingly in some embodiments step (a) comprises the steps of:

(a1) converting the compound of formula (1) into a compound of formula(Ia)

wherein Lv is a group that can be displaced by a nitrogen moiety in anucleophillic substitution reaction;(a2) reacting the compound of formula (1a) with a nitrogen nucleophileof the formula:

to form a compound of formula (2).

There are a number of ways in which the compound of formula (1) may beconverted into a compound of formula (1a) to form a compound where thecarboxylic acid moiety has been activated for further reaction with anucleophillic species. Thus for example one well known way of activatingcarboxylic acids is to convert them to the corresponding acid chlorideby reaction with thionyl chloride for example. In effect thistransformation replaces the OH group (which is a relatively poor leavinggroup) with the Cl group which is a relatively good leaving group. Anumber of transformations of this type are known where the ability ofthe OH portion of the carboxylic acid group to be displaced in anucleophillic substitution reaction is increased by reacting it withanother species. In one embodiment of the invention the carboxylic acidmoiety is reacted with an alcohol to form the corresponding ester whichis more readily substituted by a nitrogen moiety.

Once the group of formula (1a) has been formed it is reacted with anitrogen moiety typically a nitrogen of the formula:

These reactions may be carried out in a number of ways although it ispreferred if the amine can be used in excess to facilitate significantconversion of the starting material to the desired final product. Thechoice of nitrogen moiety to use will depend upon the desired L₁ groupin the final product and the moiety will be chosen on this basis. Anexample of a particularly useful nitrogen moiety is 1,3-diamino propane.

The compound of formula (2) is then converted to a compound of formula(3) by reaction with a suitable reagent to introduce a moiety that iscapable of binding with a biological entity. A wide number of reagentsthat are suitable for this purpose are known and the choice of reagentwill depend upon the nature of the group you wish to introduce. Once thereagent is chosen this will determine the suitable reaction solvent andconditions in each individual case.

In some embodiments the compound of formula (2) is converted to acompound of formula (3) by reacting the amine with a reagent selectedfrom the group consisting of an azide, thiosphosgene, carbon disulphideand an acid anhydride. In some embodiments the reagent is an azide. Insome embodiments the reagent is thiosphosgene. In some embodiments thereagent is carbon disulphide. In some embodiments the reagent is maleicanhydride.

Examples of compounds of formula (3) that may be produced using themethodology described above include:

or a metal complex thereof.

The formation of the metal complexes of the compounds synthesised inthis way is carried out using techniques well known in the art.

These compounds may then be further elaborated to produce compounds ofby reacting the reactive moiety with a suitable reactive element on abiological element. Thus for example where the R is a moiety capable oftaking part in a click chemistry reaction with a complementary moiety ona biological entity the R group will be chosen depending upon themoieties on the biological entity of interest.

The formation of the metal complexes of the compounds synthesised inthis way is carried out using techniques well known in the art.

As discussed above the compounds of the invention are useful as they canbind to a biological entity which can help them to be used in treatmentof the human body. The compounds of formula (3) which have been attachedto a biological entity and containing a radionuclide complexed with theligand may be used in either radiotherapy or in diagnostic imagingapplications. In each instance both therapy and diagnostic imaging willrely on the binding to the biological entity being involved infacilitating the localisation of the complex containing the radionuclidein the desired tissues or organs of the subject being treated/imaged.

Thus for example in relation to the use of the radiolabelled compoundsof formula (3) it is anticipated that these will be used by firstbinding them to a biological entity of interest followed byadministration of an effective amount of the radiolabelled compound to asubject followed by monitoring of the subject after a suitable timeperiod to determine if the radiolabelled compound has localised at aparticular location in the body or whether the compound is broadlyspeaking evenly distributed through the body. As a general rule wherethe radio labelled compound is localised in tissue or an organ of thebody this is indicative of the presence in that tissue or organ ofsomething that is recognised by the particular molecular recognitionmoiety used.

Accordingly judicious selection of a biological entity to connect thecompound of formula (3) to is important in determining the efficacy ofany of the radiolabelled compounds of the invention in diagnosticimaging applications. In this regard a wide range of biological entitiesthat can act as molecular recognition moieties are known in the artwhich are well characterised and which are known to selectively targetcertain receptors in the body. In particular a number of biologicalentities that can act as molecular recognition moieties or molecularrecognition portions are known that target tissue or organs when thepatient is suffering from certain medical conditions. Examples ofbiological entities that can act as molecular recognition moieties ormolecular recognition portions that are known and may be used in thisinvention include Octreotate, octreotide, [Tyr³]-octreotate,[Tyr¹]-octreotate, bombesin, bombesin(7-14), gastrin releasing peptide,single amino acids, penetratin, annexin V, TAT, cyclic RGD, glucose,glucosamine (and extended carbohydrates), folic acid, neurotensin,neuropeptide Y, cholecystokinin (CCK) analogues, vasoactive intestinalpeptide (VIP), substance P, alpha-melanocyte-stimulating hormone (MSH).For example, certain cancers are known to over express somatostatinreceptors and so the molecular recognition moiety may be one whichtargets these receptors. An example of a molecular recognition moietiesor molecular recognition portions of this type is [Tyr³]-octreotate.Another example of a molecular recognition moieties or molecularrecognition portions is cyclic RGD which is an integrin targeting cyclicpeptide. In other examples a suitable molecular recognition moieties ormolecular recognition portions is bombesin which is known to targetbreast and pancreatic cancers.

The monitoring of the subject for the location of the radiolabelledmaterial will typically provide the analyst with information regardingthe location of the radiolabelled material and hence the location of anymaterial that is targeted by the molecular recognition moiety (such ascancerous tissue). An effective amount of the compounds of the inventionwill depend upon a number of factors and will of necessity involve abalance between the amount of radioactivity required to achieve thedesired radio imaging effect and the general interest in not exposingthe subject (or their tissues or organs) to any unnecessary levels ofradiation which may be harmful.

The methods of treatment of the present invention involve administrationof a compound of formula (3) which has been bound to a suitablebiological entity and complexed to a radionuclide. The compounds offormula (3) after being bound to a biological entity are able to deliverthe radionuclide to the desired location in the body where its mode ofaction is desired. As discussed above examples of suitable biologicalentities to act as molecular recognition moieties are known in the artand a skilled artisan can select the appropriate molecular recognitionmoiety to target the desired tissue in the body to be treated.

A therapeutically effective amount can be readily determined by anattending clinician by the use of conventional techniques and byobserving results obtained under analogous circumstances. In determiningthe therapeutically effective amount a number of factors are to beconsidered including but not limited to, the species of animal, itssize, age and general health, the specific condition involved, theseverity of the condition, the response of the patient to treatment, theparticular radio labelled compound administered, the mode ofadministration, the bioavailability of the preparation administered, thedose regime selected, the use of other medications and other relevantcircumstances.

In addition the treatment regime will typically involve a number ofcycles of radiation treatment with the cycles being continued until suchtime as the condition has been ameliorated. Once again the optimalnumber of cycles and the spacing between each treatment cycle willdepend upon a number of factors such as the severity of the conditionbeing treated, the health (or lack thereof) of the subject being treatedand their reaction to radiotherapy. In general the optimal dosage amountand the optimal treatment regime can be readily determined by a skilledaddressee in the art using well known techniques.

In using the compounds of the invention they can be administered in anyform or mode which makes the compound available for the desiredapplication (imaging or radio therapy). One skilled in the art ofpreparing formulations of this type can readily select the proper formand mode of administration depending upon the particular characteristicsof the compound selected, the condition to be treated, the stage of thecondition to be treated and other relevant circumstances. We refer thereader to Remingtons Pharmaceutical Sciences, 19^(th) edition, MackPublishing Co. (1995) for further information.

The compounds of the present invention can be administered alone or inthe form of a pharmaceutical composition in combination with apharmaceutically acceptable carrier, diluent or excipient. The compoundsof the invention, while effective themselves, are typically formulatedand administered in the form of their pharmaceutically acceptable saltsas these forms are typically more stable, more easily crystallised andhave increased solubility.

The compounds are, however, typically used in the form of pharmaceuticalcompositions which are formulated depending on the desired mode ofadministration. The compositions are prepared in manners well known inthe art.

The invention in other embodiments provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. In sucha pack or kit can be found at least one container having a unit dosageof the agent(s). Conveniently, in the kits, single dosages can beprovided in sterile vials so that the clinician can employ the vialsdirectly, where the vials will have the desired amount and concentrationof compound and radio nucleotide which may be admixed prior to use.Associated with such container(s) can be various written materials suchas instructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, imaging agents or biological products, which noticereflects approval by the agency of manufacture, use or sale for humanadministration.

The compounds of the invention may be used or administered incombination with one or more additional drug(s) that are anti-cancerdrugs and/or procedures (e.g. surgery, radiotherapy) for the treatmentof the disorder/diseases mentioned. The components can be administeredin the same formulation or in separate formulations. If administered inseparate formulations the compounds of the invention may be administeredsequentially or simultaneously with the other drug(s).

In addition to being able to be administered in combination with one ormore additional drugs that include anti-cancer drugs, the compounds ofthe invention may be used in a combination therapy. When this is donethe compounds are typically administered in combination with each other.Thus one or more of the compounds of the invention may be administeredeither simultaneously (as a combined preparation) or sequentially inorder to achieve a desired effect. This is especially desirable wherethe therapeutic profile of each compound is different such that thecombined effect of the two drugs provides an improved therapeuticresult.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of micro-organisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents that delay absorptionsuch as aluminium monostearate and gelatin.

If desired, and for more effective distribution, the compounds can beincorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes, and microspheres.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

If desired, and for more effective distribution, the compounds can beincorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes, and microspheres.

The active compounds can also be in microencapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminiummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

As discussed above, the compounds of the embodiments may be useful fortreating and/or detecting proliferative diseases. Examples of such cellproliferative diseases or conditions include cancer (include anymetastases), psoriasis, and smooth muscle cell proliferative disorderssuch as restenosis. The compounds of the present invention may beparticularly useful for treating and/or detecting tumours such as breastcancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, headand/or neck cancer, or renal, gastric, pancreatic cancer and braincancer as well as hematologic malignancies such as lymphoma andleukaemia. In addition, the compounds of the present invention may beuseful for treating and/or detecting a proliferative disease that isrefractory to the treatment and/or detecting with other anti-cancerdrugs; and for treating and/or detecting hyperproliferative conditionssuch as leukaemia's, psoriasis and restenosis. In other embodiments,compounds of this invention can be used to treat and/or detectpre-cancer conditions or hyperplasia including familial adenomatouspolyposis, colonic adenomatous polyps, myeloid dysplasia, endometrialdysplasia, endometrial hyperplasia with atypia, cervical dysplasia,vaginal intraepithelial neoplasia, benign prostatic hyperplasia,papillomas of the larynx, actinic and solar keratosis, seborrheickeratosis and keratoacanthoma.

Synthesis of Compounds of the Invention

The agents of the various embodiments may be prepared using the reactionroutes and synthesis schemes as described below, employing thetechniques available in the art using starting materials that arereadily available. The preparation of particular compounds of theembodiments is described in detail in the following examples, but theartisan will recognize that the chemical reactions described may bereadily adapted to prepare a number of other agents of the variousembodiments. For example, the synthesis of non-exemplified compounds maybe successfully performed by modifications apparent to those skilled inthe art, e.g. by appropriately protecting interfering groups, bychanging to other suitable reagents known in the art, or by makingroutine modifications of reaction conditions. A list of suitableprotecting groups in organic synthesis can be found in T. W. Greene'sProtective Groups in Organic Synthesis, 3^(rd) Edition, John Wiley &Sons, 1991. Alternatively, other reactions disclosed herein or known inthe art will be recognized as having applicability for preparing othercompounds of the various embodiments.

Reagents useful for synthesizing compounds may be obtained or preparedaccording to techniques known in the art.

Synthetic procedures for the synthesis of selected compounds of formula(I) are detailed below.

Examples

In the examples described below, unless otherwise indicated, alltemperatures in the following description are in degrees Celsius and allparts and percentages are by weight, unless indicated otherwise.

Various starting materials and other reagents were purchased fromcommercial suppliers, such as Aldrich Chemical Company or LancasterSynthesis Ltd., and used without further purification, unless otherwiseindicated. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) werepurchased from Aldrich in SureSeal bottles and used as received. Allsolvents were purified by using standard methods in the art, unlessotherwise indicated. SP Sephadex C₂₅ and DOWEX 50w×2 200-400 mesh cationexchange resin was purchased from Aldrich. Fmoc-L-amino acids, HATU,HCTU and 2-chlorotrityl resin were purchased from GL Biochem Ltd(Shanghai, China). Fmoc-Lys(iv-Dde)-OH and Fmoc-D-amino acids werepurchased from Bachem AG (Switzerland). Fmoc-Pal-PEG-PS resin waspurchased from Applied Biosystems (Foster City, Calif.). Nova PEG RinkAmide resin was purchased from NovaBiochem, Darmstadt, Germany.[Co((NO₂)₂sar)]Cl₃, [Co((NH₂)₂sar)]Cl₃, (NH₂)₂sar,[Cu(NH₃)₂sar](CF₃SO₃)₄ were prepared according to establishedprocedures. (1) Geue, R. J.; Hambley, T. W.; Harrowfield, J. M.;Sargeson, A. M.; Snow, M. R. J. Am. Chem. Soc. 1984, 106, 5478-5488. (2)Bottomley, G. A.; Clark, I. J.; Creaser, I. I.; Engelhardt, L. M.; Geue,R. J.; Hagen, K. S.; Harrowfield, J. M.; Lawrance, G. A.; Lay, P. A.;Sargeson, A. M.; See, A. J.; Skelton, B. W.; White, A. H.; Wilner, F. R.Aust. J. Chem. 1994, 47, 143-179 and (3) Bernhardt, P. V.; Bramley, R.;Engelhardt, L. M.; Harrowfield, J. M.; Hockless, D. C. R.;Korybut-Daszkiewicz, B. R.; Krausz, E. R.; Morgan, T.; Sargeson, A. M.;Skelton, B. W.; White, A. H. Inorg. Chem. 1995, 34, 3589-3599.

The reactions set forth below were performed under a positive pressureof nitrogen, argon or with a drying tube, at ambient temperature (unlessotherwise stated), in anhydrous solvents, and the reaction flasks arefitted with rubber septa for the introduction of substrates and reagentsvia syringe. Glassware was oven-dried and/or heat-dried.

Work-ups were typically done by doubling the reaction volume with thereaction solvent or extraction solvent and then washing with theindicated aqueous solutions using 25% by volume of the extraction volume(unless otherwise indicated). Product solutions were dried overanhydrous sodium sulfate prior to filtration, and evaporation of thesolvents was under reduced pressure on a rotary evaporator and noted assolvents removed in vacuo. Flash column chromatography [Still et al, J.Org. Chem., 43, 2923 (1978)] was conducted using E Merck-grade flashsilica gel (47-61 mm) and a silica gel:crude material ratio of about20:1 to 50:1, unless otherwise stated. Hydrogenolysis was done at thepressure indicated or at ambient pressure.

Mass spectra were recorded in the positive ion mode on an Agilent 6510Q-TOF LC/MS Mass Spectrometer coupled to an Agilent 1100 LC system(Agilent, Palo Alto, Calif.). Data were acquired and reference masscorrected via a dual-spray electrospray ionisation source, using thefactory-defined calibration procedure. Each scan or data point on theTotal Ion Chromatogram is an average of 9652 transients, producing 1.02scans s⁻¹. Spectra were created by averaging the scans across each peak.Mass spectrometer conditions: fragmentor: 200-300 V; drying gas flow: 7L/min; nebuliser: 30 psi; drying gas temp: 325° C.; V_(cap): 4000 V;skimmer: 65 V; OCT R_(f)V: 750 V; scan range acquired: 150-3000 m/z.

HPLC-MS traces were recorded using an Agilent Eclipse Plus C₁₈ column (5μm, 2.1×150 mm) coupled to the Agilent 6510 Q-TOF LC/MS MassSpectrometer described above. 1 μL aliquots of each sample were injectedonto the column using the Agilent 1100 LC system, with a flow rate of0.5 mL/min. Data acquisition parameters are the same as those describedabove for mass spectra, with the exception of the fragmentor (fragmentorvoltage: 100 V).

NMR spectra were recorded on a Varian FT-NMR 500 spectrometer operatingat 500 MHz for ¹H NMR and 125.7 MHz for ¹³C-NMR. NMR spectra areobtained as D20 solutions (reported in ppm), using acetone as thereference standard (2.22 ppm and 30.89 ppm respectively). Other NMRsolvents were used as needed. When peak multiplicities are reported, thefollowing abbreviations are used: s=singlet, d=doublet, t=triplet,m=multiplet, br=broadened, dd=doublet of doublets, dt=doublet oftriplets. Coupling constants, when given, are reported in Hertz.

Semi-preparative HPLC purifications were performed using an Agilent 1200Series HPLC system with a 5 mL/min flow rate. Solvent gradients andcolumn specifications are described in the examples. An automatedAgilent 1200 fraction collector collected 1-3 mL fractions and fractioncollection was based on UV-Vis detection at 214 or 220 nm, with a lowerthreshold limit between 100-400 mAU. Each fraction was analysed using MSand analytical HPLC.

Analytical HPLC traces were acquired using an Agilent 1200 Series HPLCsystem and an Agilent Zorbax Eclipse XDB-C₁₈ column (4.6×150 mm, 5 μm)with a 1 mL/min flow rate and UV spectroscopic detection at 214 nm, 220nm and 270 nm.

UV-Vis spectra were acquired on a Cary 300 Bio UV-Vis spectrophotometer,from 800-200 nm at 0.500 nm data intervals with a 300.00 nm/min scanrate.

Voltametric experiments were performed with an Autolab (Eco Chemie,Utrecht, Netherlands) computer-controlled electrochemical workstation. Astandard three-electrode arrangement was used with a glassy carbon disk(d, 3 mm) as working electrode, a Pt wire as auxiliary electrode and aAg/AgCl reference electrode (silver wire in H₂O (KCl (0.1 M) AgNO₃ (0.01M)). Scan rate: 100 mV/s, sample interval: 1.06 mV, sensitivity: 1×10⁻⁴A.

HPLC traces of radiolabelled peptides were acquired using a WatersComosil C₁₈ column (4.6×150 mm) coupled to a Shimadzu LC-20AT with asodium iodide scintillation detector and a UV-Vis detector. 100 μLaliquots of each radiolabelled sample were injected onto the column,using a flow rate of 1 mL/min.

The following examples are intended to illustrate the embodimentsdisclosed and are not to be construed as being limitations thereto.Additional compounds, other than those described below, may be preparedusing the following described reaction scheme or appropriate variationsor modifications thereof.

Example 1 CuL²Cl₂.xHCl

CuL¹(CH₃CO₂)₃.xH₂O (644 mg) was dissolved in acetic anhydride (10 mL)and the resulting blue solution taken to dryness by rotary evaporation(60° C.). The residue was redissolved in H₂O and applied to a column ofDowex 50W×2 (10 cm height, 3 cm diameter). After washing with water (100mL) and 1M HCl (100 mL) the complexes were eluted with 3M HCl. The firstmajor band was collected and the solvent removed by rotary evaporationto yield the chloride salt as a purple solid (798 mg). MS:[CuC₂₁H₄₂N₈O₄]²⁺ m/z=266.63 (experimental), 266.63 (calculated).

Example 2 L².xHCl

A solution of CuL²Cl₂.xHCl in water in a two-neck flask was deoxygenatedby purging with N₂ gas for 20 mins. Under an atmosphere of N₂ gas,sodium sulfide was added and the solution turned dark green with a blackprecipitate. The reaction was stirred overnight at room temperature.After 20 hours, suspension was filtered (Whatman Filter Paper 1) and thefiltrate diluted with 1 M HCl (200 mL) resulting in the formation of acloudy, white precipitate. This precipitate was allowed to settle for 2h before it was filtered through a Millipore Steritop™ (0.22 μm, 500 mL)filter and applied to a DOWEX 50W×2 cation exchange column (H⁺ form,10×3 cm). The column was washed with 1 M HCl solution (500 mL) (toremove Na₂S) and then slowly eluted with 4 M HCl solution (200 mL). Theeluent was evaporated to dryness under reduced pressure to give a whitesolid.

Example 3 CuL³(CH₃CO₂)₂

CuL²C₁₂.xH₂O (434 mg) was dissolved in methanol (20 mL) and the solventwas removed by rotary evaporation (50° C.). The residue was converted tothe acetate salt by anion exchange chromatography on the acetate form ofDowex 1×8. The slurry was filtered and the solvent removed by rotaryevaporation and taken to dryness. The blue residue was dissolved inmethanol before the solvent was removed by rotary evaporation and takento dryness to give a blue residue (366 mg). MS: [CuC₂₂H₄₄N₈O₄]²⁺m/z=273.66 (experimental), 273.64 (calculated).

Example 4 CuL⁴Cl₃

CuL³(CH₃CO₂)₂ (360 mg) was dissolved in 1,3-diaminopropane (5 mL) andthe solution was stirred at room temperature for 40 h. The solution wasdiluted with water and applied to a column of SP-Sephadex C-25 (30 cmheight, 3 cm diameter). After washing with water (100 mL), the complexeswere eluted with 0.3 M NaCl to yield a minor leading band of hydrolysedester and a major band of the amine product. The blue solution wasapplied to a column of Dowex 50W×2 (10 cm height, 3 cm diameter). Afterwashing with water (100 mL) and 1M HCl (100 mL) the complex was elutedwith 3M HCl. The solvent was removed by rotary evaporation and taken todryness (422 mg). MS: [CuC₂₄H₅₀N₁₀O₃]²⁺ m/z=294.67 (experimental),294.67 (calculated).

Example 5 L⁴.xCH₃CO₂H

A solution of CuL⁴Cl₃.xHCl (511 mg) in water (4 mL) in a two-neck flaskwas deoxygenated by purging with N₂ gas for 20 mins. Under an atmosphereof N₂ gas, sodium sulfide (766 mg) was added and the solution turneddark green with a black precipitate. The reaction was stirred overnightat room temperature. After 20 hours, suspension was filtered (WhatmanFilter Paper 1) and the filtrate diluted with 1 M HCl (200 mL) resultingin the formation of a cloudy, white precipitate. This precipitate wasallowed to settle for 2 h before it was filtered through a MilliporeSteritop™ (0.22 μm, 500 mL) filter and applied to a DOWEX 50W×2 cationexchange column (H⁺ form, 10×3 cm). The column was washed with 1 M HClsolution (500 mL) (to remove Na₂S) and then slowly eluted with 4 M HClsolution (200 mL). The eluent was evaporated to dryness under reducedpressure to give a white solid (413 mg). The residue was converted tothe acetate salt by anion exchange chromatography on the acetate form ofDowex 1×8. The slurry was filtered and the solvent removed by rotaryevaporation and taken to dryness. The colourless residue was dissolvedin methanol before the solvent was removed by rotary evaporation andtaken to dryness to give a colourless residue (396 mg). MS:[C₂₄H₅₁N₁₀O₃]⁺ m/z=527.42 (experimental), 527.41 (calculated).

Example 6 L⁵.xHCl

To a mixture of sodium azide in water and dichloromethane was addedtriflic anhydride at 0° C. The mixture was allowed to warm to roomtemperature and was stirred vigorously for 2.5 h. The aqueous layer wasremoved and washed with dichloromethane. The organic layers werecombined and added dropwise to a solution of L⁴.xCH₃CO₂H, K₂CO₃ andZn(CH₃CO₂)₂.2H₂O in methanol and water. The mixture was stirredvigorously for 3 h. The organic layer was removed and the aqueous layerapplied to a column of Dowex 50W×2 (10 cm height, 3 cm diameter). Afterwashing with water (100 mL) and 1M HCl (100 mL) to remove Zn²⁺, theprotonated ligand was eluted with 3M HCl. The solvent was removed byrotary evaporation to yield the chloride salt.

Example 7 L⁶.xCH₃CO₂H

To a solution of L⁴.xCH₃CO₂H in acetic acid was added maleic anhydrideand the reaction was heated at 60° C. in a water bath for 30 min beforethe solvent was removed by rotary evaporation (60° C.). Residual aceticacid was removed by azeotroping with toluene and then taken to dryness.

Example 8 L⁷.xCH₃CO₂H

To a solution of thiophosgene in chloroform was added a solution ofL⁴.xCH₃CO₂H in water and the mixture was stirred vigorously for 12 h.The aqueous layer removed, washed with chloroform and taken to dryness.

Example 9 CuL⁸Cl₂.xHCl

To a solution of [Cu(CH₃)(NH₃)sar](CF₃SO₃)₃ (0.3 g, 0.1 mmol) inanhydrous N,N-dimethylacetamide (DMA) (5 mL) was added glutaricanhydride (0.08 g, 1. mmol) and diisopropylethylamine (132 μL) wereadded and the solution was heated at 70° C. for 5 h. The reaction wasmonitored using a microcolumn of SP Sephadex C-25 cation exchange (Na⁺form) eluting with 0.05 M sodium citrate solution. The solution wascooled and water (20 mL) was added. The solution was applied to a columnof SP Sephadex C-25 cation exchange (Na⁺ form, 6×3 cm). After washingwith water, the complexes were eluted with 0.05 M sodium citratesolution to yield the major leading band as the carboxylate product anda minor band of unreacted copper complex. The major band was applied toa Dowex 50W×2 cation exchange column (H⁺ form, 10×5 cm). After thecolumn was washed with water (500 mL) and 1 M HCl solution (500 mL) andthe complex was eluted with 3 M HCl and the eluent was evaporated todryness under reduced pressure at 40° C. giving a purple residue (0.23g).

Example 10 L⁸.xHCl

A solution of CuL⁸Cl₂.xHCl (0.1 g) in water (5 mL) in a two-neck flaskwas deoxygenated by purging with N₂ gas for 20 mins. Under an atmosphereof N₂ gas, sodium sulfide (0.14 g) was added and the solution turneddark green with a black precipitate. The reaction was stirred overnightat room temperature. After 20 hours, suspension was filtered (WhatmanFilter Paper 1) and the filtrate diluted with 1 M HCl (150 mL) resultingin the formation of a cloudy, white precipitate. This precipitate wasallowed to settle for 2 h before it was filtered through a MilliporeSteritop™ (0.22 μm, 500 mL) filter and applied to a Dowex 50W×2 cationexchange column (H⁺ form, 10×3 cm). The column was washed with 1 M HClsolution (150 mL) (to remove Na₂S) and then slowly eluted with 4 M HClsolution (300 mL). The eluent was evaporated to dryness under reducedpressure to give a white solid (0.09 g). MS: [C₂₀H₄₂N₇O₃]+m/z=428.34(experimental), 428.33 (calculated). ¹H NMR (D20): 6=1.03, s, CH₃; 1.91,m, 2H; 2.35, t, ³J=7.5, 2H, CH₂; 2.45, t, ³J=7, 2H, CH₂; 3.1-3.5, broad,24H, cage CH₂. ¹³C NMR (D20, residual acetone 30.9, 215.9): 8=19.4, CH₃;21.0, 33.4, 35.4, glutarate CH₂; 37.1 46.3, 48.4, 51.8, 54.2, 57.4, cageCH₂); 177.8, 178.5, CO.

Example 11 CuL⁹(CH₃CO₂)₂

CuLCl₂.xH₂O (0.2 g) was dissolved in methanol (3 mL) and the solvent wasremoved by rotary evaporation (40° C.). The residue was converted to theacetate salt by anion exchange chromatography on the acetate form ofDowex 1×8. The slurry was filtered and the solvent removed by rotaryevaporation and taken to dryness. The blue residue was dissolved inmethanol before the solvent was removed by rotary evaporation and takento dryness to give a blue residue (0.2 g). MS: [CuC₂₁H₄₃N₇O₃]²⁺m/z=(experimental), 252.14 (calculated).

Example 12 CuL¹⁰Cl₃

CuL⁹(CH₃CO₂)₂ was dissolved in 1,3-diaminopropane and the solution wasstirred at room temperature for 40 h. The solution was diluted withwater and applied to a column of SP-Sephadex C-25 (30 cm height, 3 cmdiameter). After washing with water (100 mL), the complexes were elutedwith 0.3 M NaCl to yield a minor leading band of hydrolysed ester and amajor band of the amine product. The blue solution was applied to acolumn of Dowex 50W×2 (10 cm height, 3 cm diameter). After washing withwater (100 mL) and 1M HCl (100 mL) the complex was eluted with 3M HCl.The solvent was removed by rotary evaporation and taken to dryness.

Example 13 L¹⁰.xCH₃CO₂H

A solution of CuL¹⁰Cl₃.xHCl in water in a two-neck flask wasdeoxygenated by purging with N₂ gas for 20 mins. Under an atmosphere ofN₂ gas, sodium sulfide was added and the solution turned dark green witha black precipitate. The reaction was stirred overnight at roomtemperature. After 20 hours, suspension was filtered (Whatman FilterPaper 1) and the filtrate diluted with 1 M HCl (200 mL) resulting in theformation of a cloudy, white precipitate. This precipitate was allowedto settle for 2 h before it was filtered through a Millipore Steritop™(0.22 μm, 500 mL) filter and applied to a DOWEX 50W×2 cation exchangecolumn (H⁺ form, 10×3 cm). The column was washed with 1 M HCl solution(500 mL) (to remove Na₂S) and then slowly eluted with 4 M HCl solution(200 mL). The eluent was evaporated to dryness under reduced pressure togive a white solid. The residue was converted to the acetate salt byanion exchange chromatography on the acetate form of Dowex 1×8. Theslurry was filtered and the solvent removed by rotary evaporation andtaken to dryness. The colourless residue was dissolved in methanolbefore the solvent was removed by rotary evaporation and taken todryness to give a colourless residue.

Example 14 L¹.xHCl

To a mixture of sodium azide in water and dichloromethane was addedtriflic anhydride at 0° C. The mixture was allowed to warm to roomtemperature and was stirred vigorously for 2.5 h. The aqueous layer wasremoved and washed with dichloromethane. The organic layers werecombined and added dropwise to a solution of L¹⁰.xCH₃CO₂H, K₂CO₃ andZn(CH₃CO₂)₂.2H₂O in methanol and water. The mixture was stirredvigorously for 3 h. The organic layer was removed and the aqueous layerapplied to a column of Dowex 50W×2 (10 cm height, 3 cm diameter). Afterwashing with water (100 mL) and 1M HCl (100 mL) to remove Zn²⁺, theprotonated ligand was eluted with 3M HCl. The solvent was removed byrotary evaporation to yield the chloride salt.

Example 15 L¹².xCH₃CO₂H

To a solution of L¹⁰.xCH₃CO₂H in acetic acid was added maleic anhydrideand the reaction was heated at 60° C. in a water bath for 30 min beforethe solvent was removed by rotary evaporation (60° C.). Residual aceticacid was removed by azeotroping with toluene and then taken to dryness.

Example 16 L¹³.xCH₃CO₂H

To a solution of thiophosgene in chloroform was added a solution ofL⁴.xCH₃CO₂H in water and the mixture was stirred vigorously for 12 h.The aqueous layer removed, washed with chloroform and taken to dryness.

Finally, it will be appreciated that various modifications andvariations of the methods and compositions of the invention describedherein will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are apparent tothose skilled in the art are intended to be within the scope of thepresent invention.

What is claimed is:
 1. A compound of the formula (III):

wherein Lig is a nitrogen containing macrocyclic metal ligand; L is abond or a linking moiety; L¹ is a spacer group; R is a moiety capable ofbinding to a biological entity or a protected form thereof or a synthonthereof.
 2. A compound according to claim 1 wherein Lig is a nitrogencontaining cage metal ligand of the formula:

V is selected from the group consisting of N and CR¹; each R^(x) andR^(y) are independently selected from the group consisting of H, CH₃,CO₂H, NO₂, CH₂OH, H₂PO₄, HSO₃, CN, CONH₂ and CHO; each p isindependently an integer selected from the group consisting of 2, 3, and4; R¹ is selected from the group consisting of H, OH, halogen, NO₂, NH₂,optionally substituted C₁-C₁₂alkyl, optionally substituted C₆-C₁₈aryl,cyano, CO₂R², NHR³, N(R³)₂, R² is selected from the group consisting ofH, halogen, an oxygen protecting group, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionallysubstituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂ heteroalkyl;each R³ is independently selected from the group consisting of H, anitrogen protecting group, optionally substituted C₁-C₁₂alkyl,—(C═O)-substituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl and optionally substituted C₂-C₁₂heteroalkyl.
 3. A compound according to claim 1 wherein Lig is selectedfrom the group consisting of:

wherein R¹ is as defined in claim
 24. 4. A compound according to claim 1wherein L is a linking moiety having from 1 to 20 atoms in the normalchain.
 5. A compound according to claim 1 wherein L is a group of theformula—(CH₂)_(a)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR⁴ where R⁴ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; a is an integer selected from the group consisting of1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 6. A compound according to claim 5wherein a is an integer selected from the group consisting of 1, 2, 3,4, and
 5. 7. A compound according to claim 1 wherein L is selected fromthe group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂— and—CH₂OCH₂—.
 8. A compound according to claim 1 wherein L is —CH₂CH₂CH₂—9. A compound according to claim 1 wherein L¹ is a linking moiety havingfrom 1 to atoms in the normal chain.
 10. A compound according to claim 1wherein L¹ is a group of the formula—(CH₂)_(a)—, wherein optionally one or more of the CH₂ groups may beindependently replaced by a heteroatomic group selected from S, O, P andNR⁴ where R⁴ is selected from the group consisting of H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl; a is an integer selected from the group consisting of1, 2, 3, 4, 5, 6, 7, 8, 9 and
 10. 11. A compound according to claim 10wherein a is an integer selected from the group consisting of 1, 2, 3,4, and
 5. 12. A compound according to claim 1 wherein C is selected fromthe group consisting of —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, and —CH₂CH₂CH₂CH₂.13. A compound according to claim 1 wherein L¹ is —CH₂CH₂CH₂—
 14. Acompound according to claim 1 wherein R is a moiety capable of takingpart in a click chemistry reaction with a complementary moiety on abiological entity.
 15. A compound according to claim 14 wherein R isselected from the group consisting of —NCS, CO₂H, NH₂, an azide, analkyne, an isonitrile, a tetrazine, or a protected form thereof or asynthon thereof.
 16. A compound according to claim 1 selected from thegroup consisting of:

or a metal complex or salt thereof.
 17. A compound according to claim 1,wherein the nitrogen containing macrocyclic metal ligand is coordinatedwith a metal ion.
 18. A compound according to claim 17 wherein the metalin the metal ion is selected from the group consisting of Cu, Tc, Gd,Ga, In, Co, Re, Fe, Au, Mg, Ca, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Pt,Zn, Cd, Mn, Ru, Pd, Hg, and Ti.
 19. A compound according to claim 17wherein the metal in the metal ion is a radionuclide selected from thegroup consisting of Cu, Tc, Gd, Ga, In, Co, Re, Fe, Au, Ag, Rh, Pt, Bi,Cr, W, Ni, V, Ir, Pt, Zn, Cd, Mn, Ru, Pd, Hg, and Ti.
 20. A compoundaccording to claim 17, wherein the metal in the metal ion is aradionuclide selected from the group consisting of ⁶⁰Cu, ⁶²Cu, ⁶⁴Cu and⁶⁷Cu.